Cooling apparatus used for cryonic preservation, and corresponding operating method

ABSTRACT

The invention relates to a cooling apparatus, especially for cryogenically preserving biological samples, including a duct ( 5 ) for delivering a coolant ( 3 ) to a cooling chamber ( 1 ), a heater ( 6 ) that has an adjustable first heating performance (P 2 ) for heating the coolant ( 3 ) delivered to the cooling chamber ( 1 ), a first temperature sensor ( 8 - 10 ) for measuring the temperature (T 2 -T 4 ) in the cooling chamber ( 1 ), a second temperature sensor ( 7 ) for measuring the temperature (Ti) of the coolant ( 3 ) delivered to the cooling chamber (I), and a regulator ( 11 ) for regulating the temperature. The regulator ( 11 ) is embodied as a multiple regulator which detects several temperatures (T 1 -T 4 ) as control variables and/or adjusts several heating performances (P 1 , P 2 ) as manipulated variables. The invention further relates to a corresponding operating method.

BACKGROUND OF THE INVENTION

The invention relates to cooling equipment, especially for thecryopreservation of biological samples.

The freezing of biological samples such as, e.g., stem cells in order topreserve their vitality is known within the scope of so-calledcryopreservation. A cooling down to less than −130° C. is necessary herefor a complete preservation of vitality so that liquid nitrogen isusually used as cooling agent. However, not only the low storagetemperature is important for the preservation of vitality but also theobservance of a given temperature course in time during freezing andthawing.

In order to meet these requirements, cooling equipment is obtainable,e.g., from DE 88 07 267.3 that uses liquid nitrogen with a boiling pointof −196° C. as cooling agent. The liquid nitrogen is at first located ina cooling agent storage container and is heated in it by an electricallyoperated evaporator, the outgassing nitrogen being conducted via acooling agent supply line into a cooling chamber and correspondinglycools its inner space so that material to be cooled located in thecooling chamber is frozen.

However, the mere outgassing of nitrogen by the evaporator only makescooling agent temperatures near the boiling point of −196° C. possible,whereas, on the other hand, the cooling chamber should also be cooled tohigher temperatures, especially during the freezing and thawing.Therefore, an electrically operated heater that heats the outgassingnitrogen to the desired temperature is arranged in the cooling agentsupply line between the cooling agent storage container and the coolingchamber.

Furthermore, the known cooling equipment comprises a control device thatmeasures the temperature of the cooling agent introduced into thecooling chamber as a control variable and adjusts the heatingperformance of the heater arranged in the cooling agent supply line as amanipulated variable in order to achieve the desired temperature coursein time during freezing and thawing. Thus, the control device controlsonly a single heater and evaluates only a single temperature.

However, the previously described, known cooling equipment has thedisadvantage of an unsatisfactory control behavior, which expressesitself in an overswinging between the target temperature and the actualtemperature and results in a deviation from the desired temperaturecourse in time during freezing and thawing. As a result, theunsatisfactory control behavior of the known cooling equipment canresult in damage to the biological samples to be preserved.

The invention therefore has the task of improving the temperaturecontrol behavior in the previously described, known cooling equipment.

SUMMARY OF THE INVENTION

The invention comprises the general technical teaching of detecting notonly the temperature in the cooling chamber but also at least one othertemperature such as, e.g., the temperature of the heated cooling agentsupplied to the cooling chamber as control variables.

Furthermore, the invention also comprises the general technical teachingof adjusting at least one further manipulated variable such as, e.g.,the heating performance of the evaporator arranged in the cooling agentstorage container in addition to the heating performance of the heaterarranged in the cooling agent supply line.

Therefore, the cooling equipment of the invention preferably has amultiple controller that detects several temperatures as controlvariables and/or adjusts several heating performances as manipulatedvariables. The concept of a multiple controller used here is to beunderstood in a general manner and not limited to a single controllerthat has several inputs and/or several outputs, but it is also possiblethat the multiple controller comprises two substantially separatecontrol circuits.

Thus, for example, one control circuit can detect the temperature in thecooling chamber as a control variable and adjust the heating performanceof the evaporator as a manipulated variable while another controlcircuit detects the temperature of the heated cooling agent prior to itsintroduction into the cooling chamber as a control variable and adjuststhe heating performance of the heater arranged in the cooling agentsupply line as a manipulated variable.

If the actual temperature in the cooling chamber is above the targettemperature, the heating performance of the evaporator is increased sothat more nitrogen outgases and passes into the cooling chamber, whichresults in a correspondingly greater cooling.

On the other hand, if the actual temperature in the cooling chamber isless than the target temperature, the heating performance of theevaporator is reduced in order that less nitrogen outgases. Thisdown-regulation of the evaporator when the cooling is sufficient alsohas the advantage that nitrogen is not consumed unnecessarily.

The controlling of the heating performance of the heater arranged in thecooling agent supply line takes place in a similar manner in that thisheating performance is increased when the actual temperature of theheated cooling agent is below the target temperature in the coolingchamber. In a corresponding manner, the heating performance of theheater arranged in the cooling agent supply line is reduced if theactual temperature of the heated cooling agent is above the targettemperature in the cooling chamber.

In a preferred exemplary embodiment of the invention the measuring ofthe temperature in the cooling chamber does not take place by a singletemperature sensor but rather by several temperature sensors that arepreferably arranged in a spatially distributed manner in order to beable to detect local temperature variations within the cooling chamber.The control device can then takes into account the formation of localtemperature peaks within the cooling chamber by forming an average valueand supplying documentation about the actual temperature distribution.

It is furthermore advantageous if at least one temperature sensor has athermocouple whereas another temperature sensor is designed as atemperature-dependent electrical resistor. Such a combination ofdifferent sensor types is appropriate since in this manner theadvantages of the different sensor types can be utilized and thedisadvantages are avoided. Thus, thermocouples have a good dynamicbehavior as temperature sensors but the accuracy is relatively low. Onthe other hand, temperature-dependent electrical resistors have a poordynamic behavior due to their thermal inertia but have great accuracy.Thus, the temperature can be measured very dynamically and veryaccurately by a combination of these two sensor types.

For example, so-called NTC's (negative temperature coefficients) orPTC's (positive temperature coefficients) can be used astemperature-dependent electrical resistors.

Furthermore, the cooling equipment of the invention preferably has astorage equipment in order to record the temperature in the coolingchamber and/or the temperature of the heated cooling agent before itenters into the cooling chamber. For example, a commercial PC that isconnected via a data interface to the control device of the coolingequipment according to the invention can be used for this. Furthermore,such a PC can also assume the task of setting the desired temperaturecourses in time during freezing and thawing.

It is furthermore desirable in the cryopreservation of biologicalsamples to avoid spatial temperature fluctuations within the coolingchamber in order that a defined freezing or thawing is possibleindependent of the positioning of the biological sample to be preservedwithin the cooling chamber. In the preferred exemplary embodiment of theinvention, the cooling agent supply line therefore empties via adiffuser into the cooling chamber, the diffuser distributing the coolingagent that is streaming in as uniformly as possible in the coolingchamber. Such a diffuser can consist, e.g., of an antechamber into whichthe cooling agent is first introduced, the antechamber being connectedover a large area via outlets to the cooling chamber in order to avoidlocal temperature influences.

In a variant of the invention, the cooling agent supply line emptieslaterally and preferably only on one side of the cooling chamber intothe cooling chamber. This is advantageous since streams of coolingagents then form inside the cooling chamber that rapidly result in athorough mixing and a temperature adjustment.

In contrast thereto, in another variant of the invention the coolingagent supply line empties on the top of the cooling chamber into thecooling chamber, which can be particularly advantageous if the coolingchamber is a cooling bell open on the bottom.

The concept of a cooling chamber cited here is therefore not limited tostationary cooling chambers into which the cooled material isintroduced, but it is also possible that the cooling chamber is a mobilecooling bell that is placed on the particular cooled material.

It should also be mentioned that the invention is not limited tonitrogen as cooling agent but it is also possible within the frameworkof the invention to use other cooling agents such as, e.g., air orhelium.

In addition, the invention also comprises a corresponding operatingmethod for such a cooling equipment.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

Other advantageous further developments of the invention are explainedin detail in the following together with the description of thepreferred exemplary embodiments of the invention using the figures.

FIG. 1 shows a schematic view of a cooling equipment in accordance withthe invention for the cryopreservation of biological samples.

FIG. 2 shows an alternative exemplary embodiment of a cooling equipmentin accordance with the invention.

FIG. 3 shows a control-engineering equivalent circuit diagram of thecooling equipment in accordance with the invention.

FIG. 4 shows a temperature course in time in the cooling chamber duringthe freezing of biological samples.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The cooling equipment shown in FIG. 1 serves the vitality-preservingcryopreservation of biological samples in which the samples are frozenand thawed in a cooling chamber 1.

In addition, the cooling equipment has a cooling agent storage container2 in which liquid nitrogen is present as cooling agent 3, where thecooling agent 3 can be evaporated by an electrically operated evaporator4.

The cooling agent 3 outgassing into the cooling agent storage container2 with a temperature close to the boiling point of −196° C. then passesvia a cooling agent supply line 5 into the cooling chamber 1, whichresults in a corresponding cooling.

The evaporator 4 has an adjustable heating performance P1 in thisinstance in order to be able to vary the intensity of the cooling. Thus,a large amount of the cooling agent 3 outgasses at a high heatingperformance P1 of the evaporator, which results in a corresponding,strong cooling action. On the other hand, less of the cooling agent 3outgasses at a low heating performance P1 of the evaporator 4 so thatthe cooling action is less as well.

In addition, in order to temper the cooling chamber 1, a heater 6 withan adjustable heating performance P2 is provided, the heater 6 beingarranged in the cooling agent supply line 5 and the heating coolingagent 3 outgassing from the cooling agent storage container 2 prior toits entrance into the cooling chamber 1 in order to achieve temperaturesabove the boiling point of −196° C., in particular during the freezingand thawing.

Four temperature sensors 7-10 are provided for monitoring thetemperature, the temperature sensor 7 measuring a temperature value T1that reproduces the temperature of the cooling agent 3 heated by theheater 6 before its entrance into the cooling chamber 1.

In contrast thereto, temperature sensors 8-10 measure temperature valuesT2, T3 and T4 that reproduce the temperature inside the cooling chamber1 at different points.

The temperature sensors 8-10 are arranged in a spatially distributedmanner so that local temperature peaks in the cooling chamber 1 can becompensated by a formation of an average value.

A control device equipment 11 is provided for temperature control here,that detects temperatures T1-T4 as control variables and adjusts theheating performance P1 of the evaporator 4 and the heating performanceP2 of the heater 6 as manipulated variables in order to maintain adesired temperature course in time during freezing and thawing, wherethe temperature course can be given by a conventional PC 12 connected tothe control device 11 via a data interface. In addition, the PC 12 alsorecords the temperature values T1-T4 measured by the temperature sensors7-10 and stores them for subsequent evaluation.

It should further be mentioned that the cooling agent supply line 5 doesnot empty directly into the cooling chamber 1 but rather indirectly viaan antechamber 13 in order to avoid spatial temperature variations inthe cooling chamber 1. To this end, the antechamber has a diffuser 4 atthe transition to the cooling chamber 1 that results in a turbulence ofthe cooling agent 3 entering into the cooling chamber 1. Furthermore,the discharge cross section of the antechamber 13 is substantiallylarger at the transition to the cooling chamber 1 than the entrancecross section at the transition from the cooling agent supply line 5 tothe antechamber 13, so that the introduction of the cooling agent intothe cooling chamber 1 takes place over a relatively large area.

The temperature control behavior of the control device 11 is describedin the following using the control-engineering equivalent circuitdiagram shown in FIG. 3.

Thus, the PC 12 constantly sets a target temperature T_(TARGET), that iscompared with an actual temperature T_(ACTUAL,CHAMBER) by a subtracter20, the actual temperature T_(ACTUAL,CHAMBER) being calculated as theaverage value of temperatures T2, T3 and T4.

The subtracter 20 calculates a target-actual deviation ΔT_(CHAMBER) fromthe target temperature T_(TARGET) and the actual temperatureT_(ACTUAL,CHAMBER) and conducts it to a controller 21 thatcorrespondingly adjusts the heating performance P1 of the evaporator 4.

Furthermore, the control-engineering equivalent circuit diagram shows acontrolled system 22 that reacts to the heating performance P1 of theevaporator 4 and the heating performance P2 of the heater 6 so that theactual temperature T_(ACTUAL,CHAMBER) is adjusted.

In addition to the previously described control circuit for theevaporator 4, the control device 11 has another control circuit foradjusting the heating performance P2 of the heater 6.

Thus, the target temperature T_(TARGET) for the temperature inside thecooling chamber 1 is supplied to another subtracter 23 that compares thetarget temperature T_(TARGET) with the actual temperature T1 of theheated cooling agent. The subtracter 23 calculates a target-actualdeviation ΔT_(COOLING AGENT) from the above and supplies it to anothercontroller 24 that appropriately adjusts the heating performance P2 ofthe heater 6, whereupon the controlled system 22 reacts in anappropriate manner so that the actual temperature T1 is adjusted.

The controller 24 controls the heating performance P2 of the heater 6 insuch a manner in this instance that the actual temperature T1 of thecooling agent 3 supplied to the cooling chamber 1 corresponds to theextent possible to the target temperature T_(TARGET) in the coolingchamber 1.

The exemplary embodiment shown in FIG. 2 largely corresponds to thepreviously described exemplary embodiment shown in FIG. 1, so that inorder to avoid repetitions, reference is made to the previousdescription for FIG. 1 and the same reference signs are used in thefollowing for corresponding structural components, that arecharacterized by an apostrophe solely in order to distinguish them.

A particularity of this exemplary embodiment consists in the fact thatthe cooling chamber 1′ is designed to be open on its bottom andbell-shaped. Thus, the cooling chamber 1′ is mobile in this instance andcan therefore be placed on a biological sample 15′ to be frozen, thesample 15′ resting on a solid base 16′ such as, e.g., a laboratorytable. The cooling agent supply line 5′ is therefore flexible in thisexemplary embodiment in order to make a flexible handling of the coolingchamber 1′ possible.

Another difference of this exemplary embodiment from the exemplaryembodiment shown in FIG. 1 is that the cooling agent supply line 5′empties into the cooling chamber 1′ at the top of the cooling chamber1′.

Moreover, the cooling equipment in this exemplary embodiment can haveanother temperature sensor 17′ attached in the cooling chamber 1′ by aholding arm 18′. The holding arm 18′ positions the temperature sensor17′ inside the cooling chamber 1′ at the location at which the sample15′ is located when the cooling chamber 1′ is placed on the base 16′. Inthis manner, the temperature sensor 17′ very accurately measures thelocal temperature at the location of the sample 15′, which makes a veryaccurate temperature control possible.

Furthermore, a temperature sensor 19′ can be arranged directly on thesample 15′ or a support carrying the sample 15′ in this exemplaryembodiment, which makes an even more accurate measuring of the sampletemperature possible since local temperature variations in the coolingchamber 1′ are not taken into consideration.

The transmission of the temperature measured by the temperature sensor19′ to the control device 11′ can take place, e.g., by traditionalelectrical lines. However, it is basically also possible to transmit thetemperature measured by the temperature sensor 19′ in a wireless mannerto the control device 11′. The mobility and portability of the coolingchamber 1′ is not adversely affected by such a wireless transmission.The wireless transmission of the measured temperature can take place,e.g., by a transponder integrated in the temperature sensor 19′ or in asample carrier. There are multiple known possibilities here as regardsthe transmission type such as, e.g., radio transmission, ultrasonictransmission, optical transmission, in particular infrared transmission,etc.

Finally, FIG. 4 shows a typical temperature course in time in coolingchamber 1 during the freezing of a biological sample within theframework of cryopreservation. It is apparent from it that severalcooling and warming phases are successively passed through duringfreezing in order to freeze the biological samples while preserving asmuch vitality as possible.

However, any desired cooling and warming phases are possible within theframework of the invention, where the time of the individual phases andthe cooling and warming temperature can be set as desired.

The invention is not limited to the preferred exemplary embodimentspreviously described, but rather a plurality of variants andmodifications are possible that also make use of the concept of theinvention and therefore fall within its protective range.

LIST OF REFERENCE SIGNS

-   1, 1′ cooling chamber-   2, 2′ cooling agent storage container-   3, 3′ cooling agent-   4, 4′ evaporator-   5, 5′ cooling agent supply line-   6, 6′ heater-   7, 7′ temperature sensor-   8, 8′ temperature sensor-   9, 9′ temperature sensor-   10, 10′ temperature sensor-   11, 11′ control device-   12, 12′ PC-   13, 13′ antechamber-   14, 14′ diffuser-   15′ sample-   16′ base-   17′ temperature sensor-   18′ holding arm-   19′ temperature sensor-   20 subtracter-   21 controller-   22 controlled system-   23 subtracter-   24 controller-   P1, P1′ heating performance of the evaporator-   P2, P2′ heating performance of the heater-   T1, T1′ temperature of the heated cooling agent-   T2-T4, T2′, T3′ temperature inside the cooling chamber

What is claimed is:
 1. Cooling equipment, comprising: a) a cooling agentstorage container housing a cooling agent; b) a non-recirculatingcooling chamber; c) a cooling agent supply line connected to the coolingagent storage container for supplying the cooling agent to thenon-recirculating cooling chamber; d) an antechamber in fluidcommunication between the cooling agent supply line and the coolingchamber, said antechamber including a diffuser spatially diffusing thecooling agent directly from the cooling agent supply line into distinctchannels for supply to the cooling chamber; e) a heater with anadjustable first heating performance for heating the cooling agentsupplied to the cooling chamber, wherein the heater is integrated in thecooling agent supply line; f) an evaporator in the cooling agent storagecontainer with an adjustable second heating performance for evaporatingthe cooling agent present in the cooling agent storage container; g) afirst temperature sensor for measuring the temperature in the coolingchamber; h) a second temperature sensor for measuring an agenttemperature of the cooling agent supplied to the cooling chamber; and i)a controller for temperature control, the controller having an inputside and an output side, the input side connected to the firsttemperature sensor and the second temperature sensor, the output sideconnected to the heater and the evaporator, wherein the controller: (i)is adapted to detect several temperatures from said first temperaturesensor and said second temperature sensor as control variables; (ii) isa multiple controller; and (iii) adjusts said first heating performanceand said second heating performance as manipulated variables based onsaid temperatures detected by said first temperature sensor and saidsecond temperature sensor; wherein said multiple controller includes: afirst control circuit that detects temperature from the firsttemperature sensor in the cooling chamber as a first control variableand adjusts said adjustable second heating performance of the evaporatoras a first manipulated variable based on the first control variable; anda second control circuit that detects temperature from the secondtemperature sensor measuring temperature of the cooling agent beforeentering the cooling chamber as a second control variable and adjustssaid adjustable first heating performance of the heater as a secondmanipulated variable based on the second control variable; wherein theheater is between the evaporator and the first temperature sensor, andthe first temperature sensor controls the evaporator; and wherein thesecond temperature sensor is between the evaporator and the firsttemperature sensor, and the second temperature sensor controls theheater.
 2. The cooling equipment according to claim 1, wherein severaltemperature sensors connected to the controller are provided formeasuring the chamber temperature in the cooling chamber.
 3. The coolingequipment according to claim 2, wherein the temperature sensors arearranged in a spatially distributed manner for measuring a spatialdistribution of temperature.
 4. The cooling equipment according to claim2, wherein at least one of the temperature sensors is a thermocouple andat least one of the temperature sensors is a temperature-dependentelectrical resistor.
 5. The cooling equipment according to claim 1,wherein the cooling agent is nitrogen.
 6. The cooling equipmentaccording to claim 1, wherein the first temperature sensor and thesecond temperature sensor are connected to storage equipment that storestemperature courses.
 7. The cooling equipment according to claim 1,wherein the cooling agent supply line is adapted to empty via thediffuser into the cooling chamber.
 8. The cooling equipment according toclaim 1, wherein the cooling agent supply line is adapted to emptylaterally into the cooling chamber.
 9. The cooling equipment accordingto claim 8, wherein the cooling agent supply line is adapted to emptyinto the cooling chamber only on one side of the cooling chamber. 10.The cooling equipment according to claim 1, wherein the cooling agentsupply line is adapted to empty into the cooling chamber at the top ofthe cooling chamber.
 11. The cooling equipment according to claim 1,wherein the cooling chamber is closed.
 12. The cooling equipmentaccording to claim 1, wherein the cooling chamber is open on its bottom.13. The cooling equipment according to claim 1, wherein the coolingchamber is portable.
 14. The cooling equipment according to claim 1,wherein the first temperature sensor is arranged inside the coolingchamber and at an interval from a wall of the cooling chamber.
 15. Thecooling equipment according to claim 14, wherein the first temperaturesensor is fastened to the cooling chamber by holding equipment extendinginto the cooling chamber.
 16. The cooling equipment according to claim14, wherein the first temperature sensor is attached to a sample or to asample holder.
 17. The cooling equipment according to claim 1, whereinthe first temperature sensor is connected to a transponder thattransmits a measured temperature in a wireless manner to the controller.18. The cooling equipment according to claim 17, wherein the transponderis selected from the group consisting of a radio transponder, anultrasonic transponder, an optical transponder and an infraredtransponder.
 19. An operating method for cooling equipment, said methodcomprising: a) introducing a cooling agent from a cooling agent supplyline into a non-recirculating cooling chamber for cooling cooledmaterial, the introducing step including spatially diffusing the coolingagent directly from the cooling agent supply line into distinct channelsfor supply of the cooling agent to the non-recirculating cooling chambervia a diffuser of an antechamber in fluid communication between thecooling agent supply line and the non-recirculating cooling chamber; b)heating with a heater the cooling agent prior to the introducing stepwith an adjustable first heating performance; c) measuring an agenttemperature of the heated cooling agent prior to being supplied to thecooling chamber; d) measuring of a chamber temperature in thenon-recirculating cooling chamber; and e) controlling the agenttemperature and the chamber temperature in that both temperatures aredetected as control variables, including controlling the adjustment of asecond heating performance of said cooling agent prior to heating instep b) as another manipulated variable in addition to controlling ofthe first heating performance based on said measured agent temperatureand said measured chamber temperature; wherein step e) includes thesteps of: detecting temperature in the cooling chamber as a firstcontrol variable and adjusting heating performance of an evaporator in acooling agent storage container that supplies the cooling agent to thecooling chamber as a first manipulated variable based on the firstcontrol variable; and detecting temperature of the cooling agent afterleaving the cooling agent storage container and before entering thecooling chamber as a second control variable and adjusting the heatingin step b) as a second manipulated variable based on the second controlvariable, wherein the heating is after the cooling agent storagecontainer; wherein the heater is between the evaporator and a firsttemperature sensor that detects temperature in the cooling chamber, andthe first temperature sensor controls the evaporator; and wherein asecond temperature sensor that detects temperature of the cooling agentafter leaving the cooling agent storage container and before enteringthe cooling chamber is between the evaporator and the first temperaturesensor, and the second temperature sensor controls the heater.
 20. Theoperating method according to claim 19, further comprising the followingsteps: f) evaporating the liquid cooling agent in a cooling agentstorage container with an adjustable second heating performance toprovide an evaporated cooling agent; g) heating the evaporated coolingagent prior to the introducing step with the adjustable first heatingperformance; and h) multiple controlling of the first heatingperformance and of the second heating performance.
 21. The operatingmethod according to claim 19, further comprising the following steps: f)measuring of several spatially distributed temperatures inside thecooling chamber; and g) multiple controlling of the first heatingperformance and of the second heating performance as a function of thedifferent temperatures inside the cooling chamber.
 22. The operatingmethod according to claim 19, further comprising the following steps: f)measuring with a thermocouple the chamber temperature and the agenttemperature prior to the introducing step; g) measuring with atemperature-dependent resistor the chamber temperature and the agenttemperature prior to the introducing step; and h) multiple controllingof the first heating performance and of the second heating performanceas a function of temperatures measured by the thermocouple and oftemperatures measured by the temperature-dependent resistor.
 23. Theoperating method according to claim 19, further comprising the followingsteps: f) setting a target value in the cooling chamber, g) controllingthe agent temperature of the cooling agent entering into the coolingchamber in accordance with the target value set for the cooling chamberby adjusting the first heating performance.
 24. The operating methodaccording to claim 23, wherein the agent temperature of the coolingagent entering into the cooling chamber is controlled to the targetvalue for the chamber temperature in the cooling chamber.
 25. A methodof cryopreserving a biological sample comprising cooling the biologicalsample in the cooling equipment according to claim
 1. 26. The coolingequipment according to claim 1, wherein the second temperature sensormeasures temperature at a location before the cooling chamber and afterthe evaporator.